1. Field of the Invention
The present invention relates to an adsorbent for use in chromatography, more particularly, the present invention relates to an adsorbent which comprises hydroxyapatite particles, a method of producing the adsorbent, and a column system for use in chromatography makes use of such adsorbent.
2. Description of the Prior Art
The term "hydroxyapatite" as used herein is intended to define a chemical substance having a formula Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2 (unit cell of hexagonal system: a b=120.degree., a c=b c=90.degree., .vertline.a.vertline.=.vertline.b.vertline.=9.42 .ANG. and .vertline.c.vertline.=6.88 .ANG.) and being a species of calcium phosphates among apatite compounds belonging in turn to a hexagonal system P6.sub.3/m having a basic composition of M.sub.10 (ZO.sub.4).sub.6 X.sub.2, wherein M=Ga, Z=P and X=OH. The basic composition may also be considered as a monoclinic system P2.sub.1/b which is double the first stated basic composition. This hydroxyapatite is hereafter referred to as HAp.
These HAp particles exhibit superior properties in chromatographic separation or in substance separation or development when used as the adsorbent packed or charged into a column or as a stationary phase agent in the column in column chromatography. Thus it is possible with a chromatography using these particles as the adsorbent packed in the column, or HAp chromatography, to realize high acuteness and precision separation and fractionation of substances having a minute difference in structure from one another. This was difficult to achieve with the use of the prior-art adsorbents such as ion exchange resins, activated alumina and calcium carbonates. These substances may include biological macromolecule materials having a molecular weight of 10.sup.4 to 10.sup.9 Dalton, such as, proteins, including immunoglobulin, interferon or enzymes, or nucleic acids, such as RNA, DNA or plasmids or viruses. These are at the present time used as means that are indispensable for high purity separation and refining of a variety of ultimately useful substances obtained by gene recombination, cell fusion or cell culture en masse.
It will be noted that, for stable chromatographic separation in column chromatography having high precision and efficiency, it is essential that, in addition to the superior properties in chromatographic separation of the adsorbent particulate material packed into the column as the stationary phase, the following basic properties are required.
(a) That the sample fluid and the carrier fluid introduced into and circulated through the column, so as to flow through interstices among the packed adsorbent particles, will have a flow pattern that is as uniform as possible at any point in the transverse section of the column;
(b) and that the fluid flow rate through the column per unit sectional area and unit time is as high as possible.
The uniform fluid flow pattern at any point in the sectional column area as per item (a) above may be realized when the size of the individual adsorbent particles packed in the column is small and the extent of the particle size fluctuations is also small, the extent of the fluctuations in the size of the interstices among the packed particles at each point in the sectional plane of the column, is reduced. However, too small a size of the individual particles would result in increased resistance being presented to the fluids introduced into the column and correspondingly lowering of the fluid flow rate in the column.
Thus, for realizing a stable chromatographic separation having high precision and efficiency, it is essential that the adsorbent particles that are packed into the column exhibit superior properties of chromatographic separation, that the individual particles be of a size large enough to procure a flow level through the column which is higher than a prescribed value, and that the particle size be uniform and have only small size fluctuations.
Thus, in HAp chromatography, the following type A and type B materials are predominantly used for packings in a chromatographic column. In addition, the following type C particles have been discovered by the KOKEN Co., Ltd., the assignee of the present invention.